Increasing bacterial resistance to antibiotics and antimicrobials is a growing concern facing the medical, food, and sanitation industries (8, 11). Major mechanisms of bacterial resistance to antimicrobials include active drug efflux systems, mutations that result in altered cell permeability, cellular degradation of antimicrobials, and alterations of their cellular targets (14, 17). In efforts to counter the increasing incidence of antibiotic resistance, the pharmaceutical and food industries have invested substantial resources in the search for new inhibitory compounds of microbial, plant, and animal origin. Additionally, new synthetic and semisynthetic antibiotics that are resistant to enzymatic degradation or modification have been introduced (14, 17). Although specific mechanisms of bacterial resistance to antimicrobials have been characterized and bypassed in certain cases, the more general mechanisms of altered cellular permeability to antimicrobial agents are much less understood on a physical and molecular basis. Addressing cellular permeability barriers by using methods for enhancing the uptake of antimicrobial agents could therefore represent an important means both for decreasing antibiotic usage levels and in preventing resistance, as alterations in permeability would be expected to involve a number of genes and require modification of the structurally complex cell membrane and cell wall structures of microorganisms.

Unless noted otherwise, all chemicals used in this study were from Sigma (St. Louis, Mo.). To initially determine the ability of sesquiterpenoids to permeabilize gram-positive bacterial cells, we used a flow cytometric assay for the uptake of ethidium bromide by L. fermentum (ATCC 14931). This nonpathogenic organism was chosen for reasons of safety as a model gram-positive bacterium for live cell cytometry experiments. Ethidium bromide (FW 394.3) is a membrane-impermeant model drug with limited intrinsic fluorescence. When cells with damaged or compromised cell membranes are treated with this probe, it enters the cell, where it intercalates within double-stranded DNA and becomes highly fluorescent. Because of these properties, ethidium bromide can be a useful reporter of membrane integrity (12).

Because the flow cytometry experiments showed that sesquiterpenoids could effectively permeabilize L. fermentum to ethidium bromide, we next examined the more general ability of these compounds to enhance bacterial susceptibility to other low-molecular-weight compounds, particularly antibiotics, including ciprofloxacin, clindamycin, erythromycin, gentamicin, tetracycline, and vancomycin. The ability of sesquiterpenoids to sensitize S. aureus (ATCC 6538) to antibiotics was assessed using a disk diffusion assay. Cells were grown overnight (18 to 20 h) at 30°C in 10 ml of Trypticase soy broth (Becton Dickinson and Company, Franklin Lakes, N.J.) and diluted to a concentration of 10⁷ CFU ml⁻¹ in neutral phosphate buffer. An 0.5-ml aliquot of this suspension was combined with 4.5 ml of an overlay agar prepared from Iso-Sensitest broth (Oxoid, Ogdensburg, N.Y.) plus 0.7% agar, tempered to 50°C. Sesquiterpenoids (in ethanol, 0.5% final concentration) were added to the cell-overlay mixture, and the mixture was vortexed. After vortexing, treated cell overlays were poured over hardened Iso-Sensitest agar plates (2% agar) and the plates were swirled thoroughly in alternating clockwise and counterclockwise motions to help ensure an even distribution of sesquiterpenoids throughout the overlay. This was especially important with bisabolol. Without thorough mixing, bisabolol treatments often yielded asymmetric zones of inhibition. Antibiotic disks (BBL Sensi-Disc; Becton Dickinson and Company), were placed on hardened agar overlays, and plates were incubated at 37°C for 22 to 24 h. Diameters of zones of inhibition were measured with a ruler from the bottom of each plate. Here, as elsewhere in this work, experiments were repeated at least three times. However, absolute zone size for given antibiotic-sesquiterpenoid combinations varied slightly between independent experiments, likely as a result of limited miscibility of sesquiterpenoids in the agar overlay. Therefore, the values reported in Table ​Table11 represent averages of duplicate plates for one experiment but accurately depict the trends seen in sesquiterpenoid enhancement activity. For photography (Fig. ​(Fig.2),2), cell suspensions were combined with tempered Iso-Sensitest agar as above and 2.5 ml of the resulting mixture was overlaid onto hardened Iso-Sensitest agar plates in 60-mm-diameter petri dishes.

We also examined the enhancing activity of sesquiterpenoids with antibiotics against E. coli (ATCC 25922), which as a gram-negative bacterium possesses the additional permeability barrier of an outer membrane (OM) structure. The effect of nerolidol or farnesol on the antimicrobial activity of polymyxin B against E. coli was determined at various intervals by pour plating. Cells were grown and diluted in phosphate buffer as described above for S. aureus and treated with a 1 mM concentration of either sesquiterpenoid and/or 10 μg of polymyxin B sulfate (Sigma; 8,100 units mg⁻¹) ml⁻¹. Cell suspensions were incubated in a 37°C water bath for up to 30 min, and samples were removed at appropriate intervals, diluted in the same buffer, and enumerated by pour plating with Trypticase soy agar tempered to 50°C. Plates were incubated for 24 h at 37°C prior to counting of colonies. Cells of E. coli treated with sesquiterpenoid and polymyxin B were inactivated more rapidly than were cells treated with polymyxin alone. Figure ​Figure33 illustrates this enhancing activity for nerolidol. Under the conditions of this assay, sesquiterpenoids alone had no effect on the viability of this organism.